GENETIC POLYMORPHISM OF SOME MEDICINAL BELONGING TO USING MOLECULAR MARKERS

RASHA M. A. KHALIL1, KH. A. SOLIMAN2, NAHED A. K. F. RASHED1 AND S. A. IBRAHIM2

1. Genetic Resources Dept., Research Center, Matariya, Cairo,

2. Dept. of Genetics, faculty of Agriculture, Ain Shams Univ., Cairo, Egypt

xploitation of genetic variability is diversity within and between species E of major importance in basic should have high priority. Traditionally, genetic studies and in plant improvement markers based on morphological differ- programs. The Cruciferae (Brassicaceae) ences among individuals have been used is the largest family of the to demonstrate the genetic variability, but order. It is a natural family of major with the development of electrophoretic economic importance. The number of techniques, the biochemical analysis genera about 365 and the number of become the cheapest and simplest species about 3250, Mabberley (1997) methods that offer sufficient information and Judd et al. (1999) recorded 419 to use in plant breeding and serve as a genera and 4130 species belonging to this starting point for DNA-based studies family and is classified into 13 tribes. In (Popov et al., 2002). Moreover, the best Egypt, Teckholm (1974) recorded 61 molecular markers for genome mapping, genera and 106 species distributed in marker assisted selection, phylogenetic different habitat types. On the other hand, studied, and crop conservation has low El-Hadidi and Fayed (1995) recorded 55 cost and labor requirements and high genera and 108 species for it. reliability is called inter simple sequence repeat (ISSR), has been available Mustard plants or their oils can (Zietkiewicz et al., 1994). Microsatellites heal rheumatic pains and as emetic in are very short (usually 10-20 base pair) cases of poisoning. It may be used as stretches of DNA randomly and widely stimulant for the heart (Naim et al., 1984). distributed along the genome and they can Brassica are major oil crop and broccoli, be analyzed efficiently by the polymerase cabbage and mustard are important part of chain reaction (PCR), using specific human diet worldwide. In general, genetic primers (forward and reverse) to their improvement of crops can be accelerated flanking regions. The variation in (PCR) when broad genetic diversity and the product length is a function of the number information of these genetic resources are of SSR units. Primers based on a repeat available. The collection of these genetic sequence, such as (CA)n, can be made resources and the assessment of genetic with a degenerate 3'-anchor, such as (CA)

______Egypt. J. Genet. Cytol., 39: 41-55, Jan., 2010 42 RASHA M. A. KHALIL et al.

8 RG or (AGC) 6 TY (Tsumara et al., 2- DNA extraction 1996; Nagaoka et al., 1997). ISSR Genomic DNA was extracted from markers were successfully used for fresh young leaves of ten plants chosen estimating the genetic diversity in several randomly for each population by CTAB crops, for instance barley (Afiah et al., method of Doyle and Doyle (1987). 2007a) and canola (Afiah et al., 2007b; Leaves of eleven plants from a single Afiah and Farag, 2008). In this study population were bulked prior to isozyme and ISSR were carried out on 11 extraction. DNA was quantified by ecotypes of the Cruciferae collected from spectrophotometer and gel electrophore- different localities of Egypt and to assess sis. the genetic diversity among them. 2-2- ISSR -PCR MATERIALS AND METHODS ISSR-PCR reactions were Materials of the 11 taxa were conducted using nine specific primers, for collected from various habitats in Egypt the nine genotypes as presented in Table (Table 1). (3). The reaction condition was optimized and mixturesconsisted of dNTPs (8mM 1- Extraction of total proteins mix), 2.5 µl Taq DNA polymerase (5U/ µl) 0.3 µl, 10 x buffer with 1.5 mM

Bulked leaf sample (0.25) g of MgCl2 3.0 µl, primer (l0 mM) 2.0 µl, each ecotype was ground with liquid template DNA (50 ng/µl) 2.0 µl and H2O nitrogen and mixed with extraction buffer (dd) up to 30 µl. Amplification was pH7.5 (50 mM tris, 5% glycerol and 14 carried out in Stratgene Robocycler mMB- mercapto ethanol) in a morter with Gradient 96 which was programmed for pestle, left overnight then vortexed for 15 45 cycles as follows. Denaturation (one sec and centrifuged at 10.000 rpm at 4°C cycle) 94°C for 2 minutes, followed by 30 for 10 min. The supernatants were cycles as follows 94°C for 30 second, transferred to new eppendorf tubes and 44°C for 45 sec. 72°C for 1 minute and 30 kept at -20°C until use for electrophoresis sec. and finally one cycle extension at analysis. 72°C for 20 minutes, and 4°C (infinitive). 15 µl of PCR- products were resolved in 1-1- Isozyme detection 1.5°/o GTG agarose gel electrophoresis with 1x TAE running buffer. The run was The gels were stained for the performed at 80 V for 180 min and the gel isozymes detection according to the was stained with ethidium bromide. A enzyme system and incubated at 37°C in marker of 1 Kb plus DNA Ladder 1 µg dark for complete staining after adding /µl (Invitrogen) that contains a total of the appropriate substrate and staining twenty bands ranging from 12000 to 100 solutions (Wendle and Weeden, 1990). bp was used. Bands were detected on UV- GENETIC POLYMORPHISM OF SOME MEDICINAL PLANTS 43 transilluminator and photographed by Gel (2003) detected genetic diversity for documentation system UVP2000. esterases in Brassica members and Acacia, which agreed with our results. RESULTS AND DISCUSSION Generally, β esterase demonstrated a highly polymorphism among the studied 1. Isozyme analysis species. Results indicated that this pattern revealed a total of seven bands. The first Five isozyme systems including α and second bands were monomorphic, and β Est (esterases), Prx (peroxidase), while the other six bands were Acp (acid phosphatase), Adh (alchol polymorphic. dehydrogenase), Mdh (malate dehydro- genase) were used to test the genetic Moreover, Acid phosphatase variability among the studied species (Fig. (Acph) patterns of all studied samples 1). As can be seen from the results (Table revealed a high polymorphism in band's 2) a total of 26 bands were detected using number among the studied samples, four the five isozyme systems, seven bands bands (2, 4, 5 and 6) were polymorphic, were monomrphic and the other 19 bands and two bands (1 and 3) were were polymorphic among all studied monomorphic. Tso and Chen (1997) Brassicaceae species. Isozymes have results agreed with our results and been used successfully to evaluate genetic indicated that acid phosphatase is dimeric variability within and among Brassica- isozyme which universally occurs in the ceae species (Ozaki et al., 2000; Sabu et leaves, stems, and roots of most plants. In al., 2001). Brassicaceae, it controls the transforma- tion of qiunones as secondary compounds. Esterases (Est), generally, α- esterase exhibited highly polymorphic In addition, peroxidase patterns of patterns among the studied genotypes. Est all studied samples revealed a high is a gene family hydrolyze ester bond in polymorphism in band's number among lipid to produce plant energy for the studied samples three bands (2, 4 and biochemical reactions, to allivate energy 5) were polymorphic while two were level for other metabolisms especially monomorphic bands (no.1 and 3). Our under drought conditions (Durate et al., results agreed withthose of Mara et al. 2007). Results indicated that esterase (1999) in sunflower and Lagrimini and revealed a total of seven bands, four polymorphic bands (no. 4, 5, 6 and 7) and Rothstein (1987) in tobacco who three monomorphic bands (no.1, 2 and 3). concluded that peroxidases catalyze H2O2 However, Kumar and Gupta (1985) dependent oxidation of a wide variety of ascertained that esteras patterns were substrates like phenolic residues into cell successfully used to characterize different wall polymers as monomeric isozyme. Indian mustard genotypes. Ahmed et al. Lignification, auxin metabolism, stress 44 RASHA M. A. KHALIL et al. response and defense against pathogens. two locations, Diplotaxis acris, Diplotaxis (Welinder, 1992; Schuller et al., 1996). harraand and Raphanus sativum were clustered together. While the remaining Malate dehydrogenase (Mdh) species were clustered in the second exists in the mitochondria and cytoplasm. cluster as shown in Fig. (3). It catalyzes the reversible oxidation of malate to oxaloacetate. Thus, it plays a 2. ISSR analysis major role in central metabolism. Mdh Fourteen preselected ISSR primers banding patterns gave a total of two bands were used in the present study to identify as monomorphic bands. The result agrees eleven species of Brassicaceae as shown with Mark and William (1989) and Duart in Tables (3 and 4) and Fig. (2). Fifty two et al. (2007) who indicated that Mdh in monomorphic and 71 polymorphic dis- Cactaceae and Broccoli is a dimeric tinct fragments (57.7% polymorphism) enzyme of three bands with two or three were revealed in the eleven tested species loci. with these primers. The results showed Alcohol dehydrogenase (Adh) that primers A17898, IS-O5, IS-O12 and remove two hydrogen atoms from the B17898 were highly polymorphic (75% substrate and passes them to a suitable and 85.7% polymorphism). Moreover, coenzyme acceptor like NAD+ or primers B17899, HB12, IS-O2, IS-O6, IS- NADP+ in reverse reactions. The banding 15 and IS-O4 were moderately polymor- patterns of a total of two bands were phic (from 50% to 66.6% polymorphism). monomorphic for the studied samples. On the other hand, primers IS-O1, HB10, The results agreed with these of A17899 and HB15 showed the lowest Mukhlesur et al. (2005) who concluded polymorphism (from 25% to 44.4%). The that Adh gene family has two or three loci overall results of fourteen primers were in angiosperm species and multiple loci in illustrated in Table (3). Primers IS-O4, IS- monocots. O5, HB12, HB15, IS-O6, B17899, A17899 and A17898 gave the highest Based on isozyme marker, similar- total number of bands from 9 to 12 bands. ity matrix was developed by SPSS While, primers ISO-1, B17898, B17899, computer package system in (Table 5). HB10, IS-O2, and IS-O15 gave the lowest The closest relationship was scored total number of bands from 6 to 8 bands. between Eruca sativa from two locations In general, the result indicated that ISSR with similarity of 99.9%. While Raphanus markers gave adequate distinction among sativum and Eruca sativa gave the lowest Brassicaceae species. similarity of 17% which were considered distantly related species. The dendrogram Species-specific markers based on cellective isozyme pattern separated the ten species into two main Some specific markers for some clusters. Moreover, Eruca sativa from Brassicaceae species across ISSR GENETIC POLYMORPHISM OF SOME MEDICINAL PLANTS 45 analysis are listed in Table (4). Eight out and Raphanus sativum were clustered of 123 ISSR markers were found to be together. Another subcluster but in the species specific. These markers were same cluster included Eruca sativa from scored for the presence of unique bands two locations (Samy et al., 2007). While for a given species. Brassica nigra had remaining species were clustered in the two unique bands as revealed by A17898 second cluster as shown in Fig. (4). and IS-O15. Then, the remaining species could be distinguished by one band for Genetic relationships among each marker for Anastatica herontica, Brassicaceae species Eruca sativa from Burg EL-Arab, Diplotaxis acris, D. harra, Capsella brusa The closest relationship was scored and Matthiola inaca. Eight out of fourteen between Diplotaxis acris and Diplotaxis primers gave specific markers. The results harra with similarity of 98.9%, then Eruca agreed with those of Pharmawati et al. sativa from two locations with similarity (2005) who reported that 17 out of 584 98.6%. While Eruca sativa and Brassica ISSR markers were found to be cultivar- nigra gave the lowest similarity of 9.5% specific; Hassan (2005) who found that 20 which were considered distantly related out of 110 ISSR markers were specific species. The dendrograms based on isozyme and ISSR data (Fig. 5), clearly markers among ten genotypes of Moringa indicated that there was correlation species using 11 ISSR primers and between the eleven samples of Brassica- finally, Abdel-Tawab et al. (2007) who ceae species by clustering them together showed that 48 out of 164 ISSR markers and gave us another dimension to detect were specific markers among seven the genetic variability by showing the genotypes of Mentha and Ocimum accurate differentiations and demon- species using 10 ISSR primers. strated significant levels of variation. In Based on ISSR marker, similarity this study, the dendrogram separated Eruca sativa from two locations in matrix was developed by SPSS computer subcluster, Diplotaxis harra, Diplotaxis package system in Table (6). The closest acris and Raphanus sativums clustered relationship was scored between together in another subcluster, on the Diplotaxis harra and Diplotaxis acris other hand, Brassica nigra and B. alba with similarity 99.9%. While Capsella clustered together and split away from brusa and Diplotaxis acris gave the Eruca and Diplotaxis genera into another lowest similarity of 50.3% which were second cluster, also Zilla spinosa and considered distantly related species. The Matthiola inaca were observed to share a dendrogram based on ISSR separated the separate subcluster with Capsella brusa, ten species into two main clusters. Anestatica heronticum (Muminovic et al., Moreover, Eruca sativa from two 2005; Javidfar et al., 2006; Geng et al., locations was separated in subcluster and 2007; Javidfar et al., 2007; Liu et al., Diplotaxis acris, while Diplotaxis harra 2007; Afiah et al., 2007b). 46 RASHA M. A. KHALIL et al.

SUMMARY REFERENCES

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Table (1): Taxa of Brassicaceae samples. No. Taxa Source 1 Eruca sativa Burg EL -Arab 2 Eruca sativa North Sinai 3 Diplotaxis acris Cairo- Suez Desert Road 4 Diplotaxis harra Cairo- Suez Desert Road 5 Raphanus sativum Burg EL- Arab 6 Capsella- brusa Burg EL- Arab 7 Anastatica herontica Cairo- Suez Desert Road 8 Brassica alba Cairo- Alex. Desert Road 9 Matthiola inaca Burg EL- Arab 10 Zilla spinosa Cairo- Alex. Desert Road 11 Brassica nigra Cairo- Alex. Desert Road

50 RASHA M. A. KHALIL et al.

Table (2): Polymorphism percentages generated by five isozyme systems in the eleven Brassicaceae samples. No. of mono- No. of poly- Total Type of isozyme morphic bands morphic bands bands α-Est 3 4 7 β-Est 3 4 7 Prx 2 3 5 Acph 2 4 6 Adh 2 0 2 Mdh 2 0 2 Total 14 15 29

Table (3): Nucleotide sequences and polymorphism of the ISSR primers among Brassicaceae species. No. of Primer No. of % Primer monomor Unique Total Code polymorp polymorph Sequences phic bands bands No. hic bands ism bands A17898 (CA)6 AC 3 9 1 12 75.0% B17898 (CA)6 GG 1 6 1 7 85.7% A17899 (CA)6 AG 8 4 1 12 33.3% B17899 (CT)8 TG 5 5 1 10 50.0% HB10 (GA)6 CC 6 2 0 8 25.0% HB12 (GT)8 CC 4 5 0 9 55.6% HB15 (GTG)3 GC 5 4 1 9 44.4% IS-O1 TTT (TCC)5 4 2 1 6 33.3% IS-O2 TTT (TCC)5 3 4 0 7 57.1% IS-O4 CAT (CA)7 T 3 6 0 9 66.6% IS-O5 ATTA (CA)7 2 7 0 9 77.8% IS-O6 (GA)8 CG 4 6 1 10 60.0% IS-O15 (AG)8 CT 3 5 1 8 62.5% IS-O12 (TCC)5AC 1 6 0 7 85.7% Total 52 71 8 123 57.7%

Table (4): ISSR primers for Brassicaceae species - specific marker. Unique Primer Code No. Plant species bands A17898 Brassica nigra 1 B17898 Anastatica heronitica 1 A17899 Eruca sativa – Burg EL Arab 1 B17899 Diplotaxis acris 1 HB15 Capsella brusa 1 IS-O1 Matthiola inaca 1 IS-O6 Diplotaxis harra 1 IS-O15 Brassica nigra 1 Total 8 GENETIC POLYMORPHISM OF SOME MEDICINAL PLANTS 51

Table (5): Similarity matrix among the eleven Brassicaceae samples based on isozyme marker. 1 2 3 4 5 6 7 8 9 10 2 .986 3 .489 .489 4 .486 .489 .989 5 .405 .405 .405 .529 6 .143 .143 .340 .340 .340 7 .207 .207 .207 .193 .193 .193 8 .340 .340 .397 .397 .397 .397 .633 9 .095 .095 .193 .193 .193 .857 .853 .633 10 .193 .193 .246 .246 .246 .486 .633 .849 .633 11 .095 .095 .286 .286 .286 .930 .930 .714 .930 .571

Table (6): Similarity matrix among the eleven Brassicaceae samples based on ISSR. 1 2 3 4 5 6 7 8 9 10 2 .974 3 .970 .972 4 .974 .974 .999 5 .970 .978 .980 .998 6 .513 .513 .503 .563 .591 7 .550 .575 .563 .563 .998 .984 8 .585 .566 .573 .573 .516 .979 .976 9 .550 .550 .560 .560 .573 .989 .996 .979 10 .504 .589 .589 .560 .560 .973 .979 .998 .979 11 .506 .506 .573 .505 .505 .979 .987 .979 .998 .979

Table (7): Similarity matrix among the Brassicaceae samples based on ISSR and isozyme. 1 2 3 4 5 6 7 8 9 10 2 .986 3 .489 .489 4 .486 .489 .989 5 .405 .405 .405 .529 6 .143 .143 .340 .340 .340 7 .207 .207 .207 .193 .193 .193 8 .340 .340 .397 .397 .397 .397 .633 9 .095 .095 .193 .193 .193 .857 .853 .633 10 .193 .193 .246 .246 .246 .486 .633 .849 .633 11 .095 .095 .286 .286 .286 .930 .930 .714 .930 .571

52 RASHA M. A. KHALIL et al.

1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11

β-EST α-EST

1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11

Pert Mdh

1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11

Adh Acph Fig. (1): Zymogram of β-Est, α-Est, Perx, Mdh, Adh and Acph banding patterns among the eleven Brassicaceae samples.

GENETIC POLYMORPHISM OF SOME MEDICINAL PLANTS 53

HB10 HB12

Fig (2): DNA polymorphism using ISSR with primers A17898, B17898, A17899, B17899, HB10, HB12, HB15, IS-O1, IS-O2, IS-O4, IS-O5, IS-O6, IS-O12 and IS-O15 in the 11 taxa of Brassicaceae. 54 RASHA M. A. KHALIL et al.

Zilla spinosa Matthiola inaca

Capsella brusa Anastatica herontica Brassica ngira Brassica alba Eruca sativa-North Sinai Eruca sativa-Borg EL-Arab

Diplotaxisharra Diplotaxis acris Raphanus sativum

Fig. ( 3): Dendrogram based on isozyme of Brassicacceae species.

Zilla spinosa Matthiola inaca

Capsella brusa Anastatica herontica

Brassica ngira

Brassica alba

Eruca sativa-North Sinai Eruca sativa-Borg EL-Arab Diplotaxisharra Diplotaxis acris Raphanus sativum

Fig. (4): Dendrogram based on ISSR of Brassicacceae species.

GENETIC POLYMORPHISM OF SOME MEDICINAL PLANTS 55

Zilla spinosa Matthiola inaca Capsella brusa Anastatica herontica Brassica ngira

Brassica alba

Diplotaxisharra Diplotaxis acris Raphanus sativum

Eruca sativa-North Sinai Eruca sativa-Borg EL-Arab

Fig. (5): Dendrogram based on ISSR and isozyme of Brassicacceae species.